|Year : 2013 | Volume
| Issue : 1 | Page : 32-36
Permeability of rat oral mucosa to areca nut and paan masala extracts: An experimental study
Neethu Roberts, Venkatesh V Kamath, Krishnanand Satelur
Department of Oral Pathology, Dr. Syamala Reddy Dental College, Munnekolala, Marathalli, Bangalore, India
|Date of Web Publication||20-Jun-2013|
Venkatesh V Kamath
Department of Oral Pathology, Dr. Syamala Reddy Dental College, Munnekolala, Marathalli, Bangalore - 560 037
Source of Support: None, Conflict of Interest: None
Background: The action of ingredients of areca nut, when consumed, depends on their ability to permeate the oral mucosa. The contents of the ingested substances and the barrier functions of the oral mucosa ultimately determine the flow rate of the permeability. The presence of saliva as a medium of dilution has a contributory role. The permeability coefficient of the substances will determine the intensity of the changes brought about in the tissues and resultant development of premalignant and malignant diseases. Aims and Objectives: The present study aims to evaluate and compare the permeability of the oral mucosa to areca nut and paan masala extracts (a concoction of areca nut powder with specified and non-specified additives) by stereo and light microscopy using an animal model. Materials and Methods: Sprague and Dowley (SD) rat buccal mucosa were incubated in aliquots of 20 μl at 37°C in a specially designed humidifying chamber with prepared solutions of areca nut and paan masala according to established procedures. The extent of penetration of the solutions was evaluated at 24, 48 and 72 h; using the light and stereomicroscopy. Results: Areca nut solution showed aggressive permeation and diffused the whole length of the tissues in the first 24 h peaking at 72 h where no further diffusion was noted. Paan masala extracts showed a gradual increase also peaking in 72 h. The results show that the areca nut extracts has better penetration potential than the paan masala solutions. Conclusion: Both paan masala and areca nut extracts are deleterious to the oral mucosa, but the penetration potential of areca nut extract is more than paan masala, which is probably reflective of its capacity to induce fibrotic changes in the tissues.
Keywords: Areca nut extract, paan masala extract, perfusion kinetics, permeability, rat buccal mucosa
|How to cite this article:|
Roberts N, Kamath VV, Satelur K. Permeability of rat oral mucosa to areca nut and paan masala extracts: An experimental study. J Orofac Sci 2013;5:32-6
|How to cite this URL:|
Roberts N, Kamath VV, Satelur K. Permeability of rat oral mucosa to areca nut and paan masala extracts: An experimental study. J Orofac Sci [serial online] 2013 [cited 2020 Feb 25];5:32-6. Available from: http://www.jofs.in/text.asp?2013/5/1/32/113691
| Introduction|| |
Chewing of paan masala has gained immense popularity in the Indian subcontinent over the past two decades. With the increased product promotion through mass media, paan masala is popular not just among men, but also among women and children who generally refrain from tobacco use in any form. 
The adverse effects of paan masala and areca nut on the oral cavity depend largely on the permeability of the oral mucosa to these agents. Arecaidine, the hydrolyzed by product of arecoline in areca nuts is known to induce changes in human mucosa through diffusion and penetration of mucosal barriers. 
The rat buccal mucosa has been regularly evaluated for permeability studies and has proven to be an effective model in the evaluation of various agents.  The histological and species similarity of rat mucosa to human mucosa permits extrapolation of results with an acceptable degree of confidence.
The present study is an evaluation of the permeability of the oral mucosa to paan masala and areca nut extracts using an animal model. It also aims to compare the penetration potential between the two solutions and to evaluate the results using an image analyzer (Biolux® Image analysis software).
| Materials and Methods|| |
Buccal mucosal tissues including skin, with a uniform thickness of 5 mm were collected from Sprague and Dowley (SD rats). The rats were part of an on-going study assessing effects of areca nut and paan masala being conducted by the department. Local approval by the Institutional ethics committee and by the Institutional Animal Ethics Committee (IAEC) of the animal lab was obtained prior to commission of the study. A total of 15 tissue samples were then subjected to solutions prepared from extracts of paan masala and Gutka respectively.
Preparation of the test solution
A 30% solution of areca nut and paan masala was prepared by dissolving finely powdered areca nut and paan masala in distilled water, followed by centrifuging the solutions at 15000 rpm for 30 min. The supernatant was collected and used for application. 20 μl of each solution were applied on each sample.
The assessment of permeability was performed following standard procedures as described by Squier et al.  5 mm thick rat buccal mucosal tissues were spread over a thyroform sheet. The tissues were oriented in such a way that the solutions could be dropped from the buccal mucosal surface to evaluate the penetration toward the submucosa. 20 μl of areca nut extracts and paan masala solutions were applied on to the buccal mucosa of 15 tissues each. The tissues along with thyroform sheets were kept inside a vacuum chamber, which provided an almost equivalent oral atmosphere and incubated at 37°C [Figure 1]. Observations were made at 24, 48 and 72 h of incubation. At the end of each observation the tissues were analyzed using the stereo microscope followed by processing and embedding, and eight micron thick sections were used for light microscopic evaluation. A computerized image analysis using the Biolux® software attached to an Olympus 12 megapixel camera was performed using the stereomicroscopic and light microscopic images. To support the conclusion a statistical analysis was also performed.
|Figure 1: The specially designed permeability chamber to assess perfusion kinetics of the areca nut and paan masala extracts|
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| Results|| |
Assessing the tissue changes observed at the end of 24 h using microscopic images followed by image analysis showed the penetration of paan masala restricted to the epithelium but the areca nut extract penetrated into the full depth of the tissue [Figure 2],[Figure 3],[Figure 4],[Figure 5] and [Figure 6]. The mean image analysis values were 16.968 and 55.63 microns respectively for paan masala solution and areca nut extract. The Pearson's coefficient obtained was 0.19, indicating a significant association. Pearson's correlation coefficient values ranging from 0 to 1 denotes a positive correlation [Table 1].
|Figure 2: Stereomicroscopic image of rat buccal mucosa tissue perfused with areca nut extract solution at 24 h. Note the total perfusion of the tissue indicating aggressive permeability (dark brown areas)|
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|Figure 3: Stereomicroscopic image of rat buccal mucosa tissue perfused with paan masala extract solution at 24 h. Not the limited perfusion restricted only to epithelial and juxtaepithelial areas (compare with Figure 2) indicating gradual diffusion|
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|Figure 4: Light microscopic image of rat buccal mucosa perfused with areca nut extract at 24 h. All layers show diffusion (Original stain, no counterstain; ×10)|
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|Figure 5: Light microscopic image of rat buccal mucosa perfused with paan masala extract at 24 h. Diffusion restricted to top layers of tissue only (original stain, no counterstain; ×10)|
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|Figure 6: Light microscopic image of rat buccal mucosa perfused with paan masala extract at 72 h. Diffusion extending to all layers of tissue; but intensity lighter than areca nut specimens (original stain, no counterstain; ×10)|
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|Table 1: Penetration values of the two solutions as judged by image analyzer on light and stereomicroscopic tissue samples|
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At 48 h the depth of penetration of areca nut extract almost remained as a constant with a mean value of 55.988 microns under the image analyzer. There was a progressive penetration of paan masala into the connective tissue giving a mean depth of penetration as 45.90 microns. Statistically significant association was noted with a Pearson's coefficient of 0.443.
Only negligible increase in the depth of penetration was noted at the end of 72 h in the tissues treated with areca nut extract giving a mean value of 56.22 microns under the image analyzer. There was almost a complete penetration of the paan masala solution at 72 h reaching an equivalent depth of penetration as areca nut extract with a mean value of 53.694 microns. The Pearson's coefficient of 0.598 implies a significant statistical association.
[Figure 7] depicts an exponential real time graph of the values at the stated time intervals.
|Figure 7: Real time exponential graph of the permeability values of areca nut and paan masala extracts assessed by light- and stereo-microscopy|
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| Discussion|| |
Paan masala is a dry powdered mixture of areca nut, catechu, lime, unspecified spices and flavoring agents. Two types are commercially available, "paan masala0" without tobacco, and "Gutka," a mixture of paan masala and tobacco. Areca nut, a major constituent of paan masala has been shown to exhibit mutagenic, genotoxic and carcinogenic effects in various experimental systems.  Areca nut contains a major alkaloid "arecoline," which is capable of producing deleterious effects on the oral mucosa ranging from pre-cancers to frank cancer formation. Arecoline can produce fibroblast proliferation and collagen synthesis and also penetrate the oral mucosa to produce progressive cross linking of the collagen fibers. The consumption of paan masala and areca nut are thus linked to the development of oral submucous fibrosis, a precancerous condition that can affect any part of the oral cavity and also the pharynx. 
The effect of paan masala and areca nut are dependent on the permeability of the oral mucosa to these agents. The permeability of the oral mucosa being a complex phenomenon is attributed to the structure and pathologic status of the tissue as well as the nature of the penetrants. , The most important factor that determines the extent to which any substance will penetrate the oral mucosa is its physical and chemical properties. Molecules can pass readily than ions and the small molecules have better potential of penetration than large ones. The penetration of the ionic compounds is governed by the degree of ionization at any particular pH (the pK value). The property of an undissociated molecule, which mostly influences penetration across the mucosa, is its relative solubility or partition coefficient, in nonpolar (lipid), and polar (aqueous) solvents. Substances that can dissolve in both these solvents can pass readily across mucosa, but maximum penetration is obtained with substances having slightly preferential lipid solubility. 
The result of the present study indicates that the penetration potential (diffusion coefficient) of the areca nut extract is more than that of the paan masala solution. Areca nut, extract otherwise called arecoline, is an alkaloid soluble in water, alcohols, and ether. It has the molecular formula as C 8 H 13 NO 2, a molecular weight of 155.19 and pK value of 6.84. Arecoline due to its lipophyllic characteristics readily enters intracellular space to fulfill its effects. Commercially available product paan masala has added lime (calcium hydroxide), which helps in the slow release of the active principles (mainly arecoline) when chewed or kept inside the mouth as the arecoline is readily hydrolyzed into negatively charged arecaidine, methyl ester of arecoline having a much slower penetration across the oral mucosa.  The areca nut extract, devoid of any additives, would penetrate to the extent that its composition ingredients would permit; thus, representing the true potential of the nut to penetrate the oral mucosa. Interestingly, this observation seems to support findings of increased development of oral submucous fibrosis in patients consuming raw areca nut as compared to other forms. The consumption of areca nut in a boiled, roasted form with or without additives is also a habitual feature in many parts of the Indian subcontinent. Whether the form of preparation of the areca nut (as in boiling or roasting) would alter the permeability potential needs further studies.
The present in vitro study has certain limitations in assessment of the true penetration potential of the agents. The lack of an oral environment and the non-viability of the tissues due to detachment from the animal may alter the true penetration potential. To experimentally simulate the oral environment, tissues were incubated in a specially designed vacuum chamber. The maintenance of the environment at a standard temperature at 37°C is also known to replicate the oral environment in experimental conditions.
The extent of tissue changes produced by both paan masala and areca nut extracts depends upon the permeability of the oral mucosa to these solutions. The ability to breach the barrier of the oral mucosa and expose the tissues to the contents of the ingested agents is a primary step in the initiation of tissue changes. Thus, increased diffusion coefficient would certainly point towards early development of tissue changes, all other tissue responses being considered normal. Factors like trauma due to constant irritation from chewing the nut, additives like slaked lime, which tend to destroy the epithelial barrier due to chemical burns are also important in altering the permeability. In the clinical absence of such changes, the permeability potential is directly related to the composition of the ingredients. As such, areca nut extracts do seem to have an ability to penetrate the oral mucosa more than paan masala as suggested by our study.
| Conclusion|| |
The present study effectively demonstrates the ability of both areca nut and paan masala to diffuse through the mucosa and cause an action on the underlying tissues. The findings certainly do not indicate that paan masala is much safer than areca nut (in view of the slower diffusion rate); it only serves to emphasize the increased aggressiveness of the latter over the former in its ability to penetrate the oral mucosa. Both are proved to be deleterious to the oral mucosa, but the destructive potential of areca nut extract may be more than paan masala.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]